Enzymatically crosslinked gelatin hydrogel promotes the proliferation of adipose tissue-derived stromal cells

Preparation of gelatin hydrogels

Gelatin gel formation was initiated by mTG addition. For hydrogel preparation, gelatin powder (type A, 300 Bloom; Sigma–Aldrich, MO, USA) was weighed and dissolved in phosphate-buffered saline (PBS) at 50 °C and then sterilized as rapidly as possible through 0.22 µm filters to prevent filter blockage by the cooling gel. The mTG (Bomei, China, enzyme activity units > 100 U per gram) solution was prepared by dissolving mTG in PBS to obtain 10% (wt, weight ratio) solution and then sterilizing through 0.22 µm filters. Gelatin/mTG hydrogels were prepared by mixing a certain amount of 10% mTG solution with different concentration of gelatin solutions according to the experimental need. To determine the effects of different gelatin concentration (1%, 2%, 4%, 6%, 8%, and 10% (w/v, weight/ volume)) on the gelation time and gel strength of resultant hydrogels, and the mTG dosage was retained at 10 U/g pro (enzyme activity units per gram of protein). Here, the protein is gelatin. To determine the effects of mTG dosage (2, 5, 10, 20, and 40 U/g pro) on gelation time and gel strength of the resultant hydrogels, and the concentration of gelatin solution was maintained at 4% (w/v).

Gelation time and gel strength test

For gelation time test, 2 ml of gelatin/mTG solution was added in a transparent glass vial and incubated at 37 °C, and the onset of gelling detected through the vial inverting method was recorded as gelation time. Six repeated measurements for each type of gelatin/mTG hydrogels were performed. For the gel strength test, 6 ml of gelatin/mTG mixing solution were added into a 35 mm culture dish and incubated at 37 °C for 2 h; then, the dish was fixed on the platform of a mechanical testing apparatus (HPB, Handpi, China). The detecting probe was a flat-head stainless-steel cylinder (12.5 mm in diameter, 10 mm in height) attached to a digital pull-and-push dynamometer (HP-20 Handpi, China). The probe was loaded down onto the hydrogel at a constant rate of 1.0 mm/s at room temperature (∼24 °C). The advance was stopped when the probe front reached to a 5 mm depth from the gel surface. Meanwhile, the value of loading force was recorded automatically by a mechanical measurement software (Yueqing Handpi Instruments Co., Ltd, Zhejiang, China). The peak value of the recorded curve was acquired and converted to gel strength. Six repeated measurements for each type of gelatin/mTG hydrogels were performed.

Hydrogel degradation test

The degradation of hydrogel materials in two groups was tested. The first group consisted of cell-free hydrogels, and the second group consisted of cell-containing hydrogels. To prepare cell-free hydrogels, twelve 35 mm culture dishes were pre-weighed with an electronic balance and respectively marked. Subsequently, 2%–8% concentrations of gelatin/mTG solution were loaded into the dishes with 2 ml for each dish, and the dishes were incubated at 37 °C for 2 h. After gelling, the hydrogel-containing dishes were weighed again; 2 ml of PBS was added into each dish; and the dishes were cultured at 37 °C with 5% CO₂ for eight weeks. PBS was changed every 3 days. At different time points, three dishes were removed for testing; PBS was removed completely; and the hydrogel-containing dishes were weighed. The remaining hydrogel mass was obtained by calculating the weight variation of each dish. Finally, the degree of degradation was expressed as a percentage of the remaining hydrogel mass versus the initial hydrogel mass. To prepare cell-containing hydrogels, 2 ml of gelatin/mTG solution mixed with 5.0 × 10⁶cells was added into a 35 mm culture dish. The rest of the steps were the same as the previous methodology, except that the culture medium was changed to cell expansion medium (see ‘Primary culture of ADSCs’). Cell culture medium was replaced every 3–4 days.

Primary culture of ADSCs

Animal study was approved by the Institutional Animal Care and Use Committee (IACUC) of Sichuan University, all experiments were performed in accordance with the guidelines of IACUC of Sichuan University. Subcutaneous adipose tissue was obtained from a 150 g Sprague–Dawley rat. Harvested tissue was enzymatically dissociated using 1 mg/ml collagenase type I (Sigma, MO, USA) in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, UT, USA). Digestion was carried out under continuous agitation for 45 min at 37 °C and followed by centrifugation at 283 g for 7 min. The pelleted cells were then harvested and plated on 25 mm² cell flasks. The initial plates were denoted by Passage 0 (P0). At 24 h intervals, cultures were washed with PBS to remove contaminating erythrocytes and other non-attached cells. This procedure was repeated every 24 h for three days. The plating medium consisted of high-glucose DMEM, 15% fetal bovine serum (FBS; Invitrogen, CA, USA), and 1% penicillin/streptomycin (P/S; Hyclone, UT, USA). Cells were maintained at 37 °C with 5% CO₂ and fed two times per week. When cultures reached confluence within 5–7 days after the initial plating, the adherent cells were detached with 0.25% trypsin/EDTA and then either replated at 5.0 × 10⁴ cells/cm² or immediately used in experiments. Cultures were passaged every 3–5 days, and the culture medium was replaced by expansion medium, which consists of high-glucose DMEM, 10% FBS, and 1% P/S.

Cell viability in 2D culture

Gelatin/mTG solutions with various gelatin concentrations (2%, 4%, 6%, 8% and 10%) were prepared as described above: the solutions were added into six-well tissue culture plates (TCPs), with 2 ml placed in each well. The solutions were incubated at 37 °C for gelling for 2 h. After washing thrice with sterile PBS, the hydrogels were ready for cell 2D seeding. ADSCs were digested and centrifuged at 283 g, and the supernatant was discarded. ADSCs were then seeded on the hydrogel surface at 1.0 × 10⁵ cells per well. Cells were cultured in the expansion medium and observed daily under an inverted optical microscope (CKX41, Olympus, Tokyo, Japan). After 14 days of cultivation, live/dead staining assay was employed to assess the viability of cell populations on 2D hydrogel substrates. Samples were washed thrice in sterile PBS and incubated at 37 °C for 30 min in a solution containing 2 µM calcein—AM (Sigma, St. Louis, MO, USA) and 2 µM propidium iodide (PI; Sigma, St. Louis, MO, USA) in PBS. After incubation, samples were washed again and pictures were obtained by using an inverted fluorescent microscope (XDS30; Ningbo Sunny Instruments Co., Ltd., Zhejiang, China) equipped with a video camera (MD50; Mingmei, China).

Cell viability in 3D culture

ADSCs were prepared as described above and cell density was adjusted to 5.0 × 10⁶cells/ml. Cell suspensions were mixed with different concentrations (4%, 6%, and 8%) of gelatin/mTG solution at 1:9 volume ratio. Aliquots of 100 µl were loaded into six-well TCPs and incubated at 37 °C for 2 h. After gelling, 2 ml of culture medium was added in each well and the TCPs were incubated at 37 °C in 5% CO₂. The medium was changed every three days. Cell morphology was monitored daily under an inverted optical microscope. After two weeks of cultivation, cell viability was tested via the calcein—AM/PI assay as mentioned before, and images were captured using a confocal laser scanning microscope (A1si, Nikon, Japan).

Cell proliferation assay

ADSCs were seeded on the surface of 2–8% hydrogels (2D culture) or embedded into hydrogels (3D culture) as described above at 5.0 × 10⁴ cells per well of 24-well TCPs. Cells were cultured in expansion medium for four weeks, and medium was changed every 3 days. At each time point, cells/hydrogel constructs from three parallel samples were rinsed thrice with PBS and incubated in 100 µl of 10% Alamar Blue solution (Yeasen, China) for 3 h. Subsequently, the incubation solutions were transferred to a 96-well TCP, and fluorescence was measured with a plate reader using excitation/emission wavelengths of 530/590 nm. ADSCs seeded on TCPs served as a negative control.

Cardiac differentiation of ADSCs

To investigate the differentiation status of encapsulated stem cells, ADSCs from the third passage were encapsulated in 6% gelatin/mTG hydrogels with the method described above (‘Cell viability in 3D culture’). After forming hydrogels, encapsulated cells were divided into two groups: one group was treated with normal cell expansion medium (see ‘Primary culture of ADSCs’), and the other was treated with differentiation medium. The differentiation medium consisted of DMEM with 1% P/S supplemented with 2% horse serum (HS; Hyclone, UT, USA) and 50 µg/ml L-ascorbic acid. The medium was changed twice a week, and a 14-day culture was performed. Control groups were seeded on TCPs supplemented with cell expansion medium or with differentiation medium for the same period.

The expression of cardiac-specific genes NK2 homeobox 5 (Nkx2.5), alpha cardiac myosin heavy chain 6 (Myh6), gap junction protein alpha 1 (Gja1), and myocyte enhancer factor 2C (Mef2c) in hydrogel-encapsulated cells and in control groups were measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted using Trizol reagent (Life Technologies). Complementary DNA was synthesized from 1 mg total RNA by employing RevertAid First Strand cDNA Synthesis Kit (K1622; Thermo Scientific, Vilnius, Lithuania). Complementary DNA samples were subjected to PCR amplification using Luminaris Color HiGreen qPCR Master Mix (K0391; Thermo Scientific, Vilnius, Lithuania). The DNA sequences of primers are listed in Table 1. PCR was performed with a real-time PCR Detection System (iCycler IQ5; Bio-Rad, Hercules, CA, USA). Cycles were programmed as follows: 95 °C for 10 min, 40 cycles of 15 s denaturation at 95 °C, 30 s at an annealing temperature of 57 °C, 30 s extension at 72 °C, with a final extension at 72 °C for 10 min. The 2^−ΔΔCt method was used to evaluate relative mRNA expression levels for each target gene. The expression of the housekeeping gene β-actin was employed for internal normalization. The product size was confirmed by running 10 µl of samples on 2% agarose gel electrophoresis.

Cell migration study in a 3D culture model

The migration ability of ADSCs in 3D hydrogels was evaluated by using a specially designed model in which cells were seeded. In brief, 2–8% gelatin/mTG solutions were loaded in six-well TCPs at 2 ml volume in each well and incubated at 37 °C for 3 h. Subsequently, a 2 mm radius hole was made at the center of each hydrogel by using a punch, and 25 µl of 8% gelatin/mTG solution mixed with 1.0 × 10⁷ cells was filled into the hole. The added gelatin/mTG/cells constructs were set to solidify for 2 h and then each well was supplemented with 2 ml expansion medium. The cells were cultured at 37 °C with 5% CO₂ for 14 days, and the culture medium was changed every 3 days. Meanwhile, the gelatin/mTG/cells constructs were monitored daily to observe the cell migration from the transplanted site into the surrounding matrix. Cell migration images were captured, and cell migration distances from the hole edge to the cell outgoing front in all directions were measured via image analysis software (Image Pro Plus 6.0; Media Cybernetics, Rockville, MD, USA). Afterward, the average migration distance was calculated for statistical analysis.

In vivo implantation of hydrogel

The in vivo biological response to gelatin/mTG hydrogels was assessed at four weeks in subcutaneous SD rat models (age 6–7 weeks) with a total of four rats included in this study. Hydrogels with 4% and 8% gelatin were prepared as described above, and the hydrogels were cut into sheets of 10 mm × 10 mm × 1.5 mm size. Prior to surgery, the rats were anesthetized with 10% chloral hydrate and the areas of surgery were shaved and disinfected with iodophors. A pair of hydrogel sheets with 4% and 8% gel concentrations was surgically placed within subcutaneous pockets located on both sides of an adult rat dorsum. Three rats were implanted with hydrogels, and the last rat was subjected to surgery, but no hydrogel was implanted. The incision was covered in Betadine ointment after surgical suture and assessed for signs of infection for three days after surgery. All rats survived the surgical procedure without surgical complications. After four weeks the rats were sacrificed and the implants were harvested and sectioned at 10 µm thick with a cryosection microtome (Leica CM1950; Leica, Wetzler, Germany). The sections were fixed in 4% paraformaldehyde and processed for histological analysis using hematoxylin/eosin staining.

Statistical analysis

Data are presented as mean ± SD. Statistical analyses were performed using SPSS software (version 14.0). Statistical significance between two groups was determined by Student’s t-test. Results for more than two groups were evaluated by one-way ANOVA with least-significant difference (LSD) test. P < 0.05 was considered statistically significant.

Evaluation of gelation time and gel strength

Gelation time of the gelatin/mTG solution was evaluated at 37 °C via a bottle-invert method (Fig. 1). Depending on mTG dosage or gelatin percentage in gelatin/mTG solution, the gelation time was verified to be controllable. Decrease in either mTG dosage or gelatin percentage extended gelation time. When mTG dosage was kept constant at 10 U/g pro, the gelation time was inversely proportional to gelatin concentration (shown as Table 2). The 10% gelatin/mTG solution only took 15 s before arriving gel state. By contrast, the 1% gelatin/mTG solution retained a flow state and did not become a hydrogel.

To investigate the mechanical properties of gelatin/mTG hydrogels, we employed a digital pull and push dynamometer to evaluate gel strength. We found that gel strength is proportional to the concentration of gelatin solution. For example, gel strength increased rapidly as gelatin concentration increased from 2% to 6% and retained a relative constant strength thereafter even if increasing gelatin concentration to 10%. On the other hand, considering that gel strength may also be correlated with mTG dosage we applied a variety of mTG dosages with 4% gelatin solution to test their gelation time and gel strength (shown as Table 3). The results showed 10 U/g pro could be a compromised dosage that could bring about adequate gel strength and minimize the side effects possibly caused by excessive mTG.

Hydrogel degradation performance

Given that cells seeded in a hydrogel may secrete several proteases, such as collagenase, which could lead to hydrogel degradation, the degradation performance of gelatin/mTG hydrogel should be evaluated. The degradation test was carried out in two parallel groups: cell-free hydrogels and cell-containing hydrogels. In the test of cell-free hydrogels (Fig. 2A), the degradation rate of 2% concentration of hydrogels was inversely proportional to culture time. Within three weeks, the hydrogels lost more than half of their original mass. At the eighth week, only approximately 5.6% of original mass remained. For the other three kinds of hydrogels (4%, 6% and 8%), their degradation curves were different. Apparent degradation in these hydrogels was not observed until the eighth week. For the hydrogels with 4% concentration gelatin, 3.6% mass loss was observed

In the test of cell-containing hydrogels (Fig. 2B), we found that 2% hydrogels degraded more rapidly than cell-free hydrogels. For three weeks, nearly 80% of hydrogel mass was lost. Moreover, the 4% hydrogel also showed significant degradation. At the eighth week, nearly half of the hydrogel mass was lost. However, the 6% and 8% hydrogels did not show severe degradation. For 6% hydrogel, approximately 12% of gel mass was lost after eight weeks of incubation; meanwhile for 8% hydrogels, mass loss was less than 4%.

Cell morphological observation from 2D culture

We assessed the biocompatibility of gelatin/mTG hydrogels and its effect on cell adhesion. ADSCs were seeded on the surfaces of various hydrogels for two weeks. Cell viability was evaluated via live/dead staining assay. Cell staining showed that the vast majority of ADSCs cultured on 2%–10% gels were stained green (on behalf of living cells), and only few cells were stained red (on behalf of dead cells). This finding means that these hydrogels present good biocompatibility and are suitable for cell 2D culture. In addition, regarding cell morphology, the effects of different concentrations of hydrogel materials on cell growth and adhesion behavior were different. The cells seeded on 2% hydrogels plunged into hydrogels and grew inside owing to weaker gel strength, which led to more rounded or stick-shape cells appearing than other hydrogels (Fig. 3A). As the hydrogel concentration increased to 4%, cell shape progressively assumed more of barbed-like or caltrop-like patterns, which represented the cell pseudopodia stretching out in different directions in a 3D gel space (Fig. 3B). With further increasing hydrogel concentration (Figs. 3C and 3D), cells gradually spread out and grew in size. When the hydrogel concentration increased to 10% (Fig. 3E), the cell growth pattern on the gel was similar to that on TCP (Fig. 3F).

Cell morphological observation from 3D culture

Compared with hydrogel-surface culture, the 3D culture of embedding ADSCs into a hydrogel better mimicked in vivo cell growth. Three concentrations (4%, 6% and 8%) of hydrogels were included in 3D culture experiments. As for 2% hydrogels, owing to their relatively low gel strength and susceptibility to degradation, we did not consider the hydrogels as experimental candidates. As for 10% hydrogels, we also excluded them from the experiment because in both the hydrogel degradation test and the 2D cell culture experiment, their performances did not show apparent difference with that of 8% hydrogels. In 3D culture, we found that the encapsulated ADSCs from all tested hydrogels present good viability (Fig. 4). After two weeks of culture, the slice views photographed by laser scanning confocal microscopy showed that the cells distributed evenly on the hydrogels with numerous cell protrusions. In the synthesized 3D images, we clearly saw that the ADSCs almost filled all areas inside the hydrogels. However, with the increase in hydrogel concentrations, some of the cells residing deep in these hydrogels died because of insufficient nutrient metabolism caused by the compact hydrogel.

Evaluation of cell proliferation in 2D and 3D culture

ADSC proliferation capabilities were assessed by Alamar Blue assay. During four weeks of 2D culture, significant difference in cell growth behavior was observed (Fig. 5A). On day 2, evident differences were not observed among the cells cultured on different substrates. However, from day 4 to day 12, the cells on TCP apparently showed a higher proliferation rate compared with cells cultured on the gel surface. However, after 12 days of culture, the cell number on TCP almost linearly decreased as the cells stopped growth caused by cell contact inhibition, and some cells were detached from the TCP surface. From the 2D culture experiments we found cell proliferation ability was almost proportional to the concentration of hydrogel. We also found that cell growth on lower hydrogel concentration (2% and 4%) decreases on day 18 possibly because of degradation of these materials, which led to the loss of the corresponding mechanical support. For 8% or 10% hydrogels, the cell number remained increasing despite the cell growth rate started to slow down after three weeks of culture.

ADSC proliferation in 3D culture was carried out using Alamar Blue assay (Fig. 5B). Cellular proliferation behavior in 3D culture presented a similar situation as that in 2D culture. The hydrogels at 8% or 10% concentration shows good performance in supporting cell growth because the cell number in these hydrogels continued to increase even after four weeks of culture. Cells on hydrogels with 4% gelatin concentration exhibit fast growth rate in the first two weeks; however, cells begin to fall down from day 18 after cell seeding. The 2% hydrogels exhibit poor performance and few cells survived in hydrogels after two weeks of culture. We speculate that the 2% hydrogels present weaker mechanical properties, together with the degradation of collagenase secreted by the residing cells, leading to poor performance in supporting cell 3D growth.

Relative mRNA expression of encapsulated ADSCs

To measure the effects of gelatin/mTG hydrogel on cardiogenic differentiation of ADSCs, we evaluated cardiac markers Nkx2.5, Myh6, Gja1, and Mef2c were evaluated by real-time quantitative PCR. All values are normalized to the expression level of β-actin. The mRNA expression level of ADSCs cultured on TCPs in cell expansion medium (negative control) is indicated as “1.” Each measure of gene expression in the encapsulated ADSCs is presented as a fold change over that of negative control. The results are shown in Fig. 6. The encapsulated ADSCs in differentiation medium present the highest mRNA expression level among all detected samples; their expression levels of Nkx2.5, Myh6, Gja1, and Mef2c are 15.26 ± 2.06-, 20.80 ± 2.79-, 31.01 ± 1.62-, and 14.64 ± 0.82-fold, respectively. For the encapsulated ADSCs in normal cell expansion medium, their mRNA expression levels of Nkx2.5, Myh6, Gja1, and Mef2c are 5.56 ± 0.67-, 5.97 ± 1.22-, 15.69 ± 2.05-, and 4.60 ± 0.91-fold higher compared with negative controls, respectively. No statistical difference was found in cells cultured on TCPs in the differentiation medium compared with negative controls.

Evaluation of cell migration in 3D hydrogels

For repairing the damaged tissue and organ, cells/scaffold construct implanted into the body should be able to release cells from the scaffold into the surrounding tissue. In this study, we designed a 3D cell migration model to simulate the in vivo transplant environment. The experiments show that cell migration activities started as early as the 2nd day after in vitro hydrogel implantation and continued to the end of the experiments. Low concentration of hydrogels favors cell migration. With the increase in gel concentration, the hydrogels became dense, and the average migration distances of ADSCs encapsulated in the hydrogels were decreased. After two weeks cells in the 2% hydrogels migrated by 9.36 ± 0.58 mm with an average migration speed of about 0.67 mm/day. By contrast, in the other three hydrogels cells migrated by about 3–5 mm in the same period (Fig. 7A). The experiments also showed that the released cells from the cells/hydrogel construct maintained an extremely strong vitality and proliferative capacity during two weeks of experiments (Figs. 7B–7E).

In vivo response to gelatin/mTG hydrogels

To characterize the local tissue response to gelatin/mTG materials, we implanted 4% and 8% concentration hydrogels subcutaneously into an adult SD rat model and explanted after four weeks for analysis. Approximately 6–8 h after surgery, the rats could eat and drink, and no breathing difficulties and unusual activities were observed. Their wounds were healed without the formation of scar tissue within a week (Fig. 8A). The 8% hydrogels could be clearly observed under the skin throughout the experiment period, indicating that the hydrogels are not degraded significantly. However, the 4% hydrogels could only be observed in the first two weeks and are difficult to identify after another two weeks. We speculated that material degradation had occurred. After four weeks, the rats were sacrificed, and the implants were harvested, thus confirming our speculation. All 4% concentration samples were severely eroded, but no macroscopic signs of inflammation or toxicity were evident in the tissue surrounding the implants. As for 8% hydrogel samples, apparent evidence of capsule formation was found around the implants, and only slight degradation occurred. Hematoxylin and eosin staining of sections throughout the explanted hydrogels showed no apparent infiltration of cells into the 8% hydrogels, but a small number of cells had invaded into the 4% hydrogel samples (Fig. 8B). We can see from morphological observation, that the cells were not macrophages or inflammatory cells but may be endothelial cells instead, migrating from surrounding tissues, to form capillary networks inside hydrogels.